Detailed studies defining the complexities of replication, repair and recombination have elucidated the role of individual proteins that participate in these complicated processes. Many of the details remain to be solved. The area that has only recently begun to be attacked at the biochemical level concerns mitosis, the important biological process by which chromosomes are sorted, paired and accurately distributed to each daughter cell. During mitosis centromeres direct the movement of chromosomes. The centromere is the site at which the kinetochore forms and it is the last site by which sister chromatids remain associated prior to their separation at the metaphase-anaphase transition. Considerable evidence has accumulated indicating that mitotic checkpoint controls play an important role in regulating the cell cycle. A variety of drugs that interfere with mitotic spindle assembly block the cell cycle in mitosis. Mitotic checkpoint targets have been the focus of a number of antitumor drugs. Thus, a thorough understanding of the mechanism underlying the process of mitosis may lead to a better understanding of aberrant growth. This proposal is aimed at identifying proteins that interact with centromeric DNA isolated from Schizosaccharomyces pombe. S. pombe contains 3 chromosomes and 3 distinct centromeres. The sequence of the centromeric DNAs have been largely determined and they vary in size from about 40 to greater than 100 kb. The centromeres of S. pombe are less complex than those in higher eukaryotes (300-5000 kbp) but considerably more complex than the 125 bp region on S. cerevisiae chromosomes that functions as centromere. While a number of proteins that interact with centromeres of S. cerevisiae and mammals have been defined, virtually no proteins interacting with centromeres of S. pombe have been reported. The investigators have detected a number of proteins that bind to AT-rich regions in duplex DNA. These proteins were isolated based on their binding to origin sequences derived from autonomous recognition sequences (ARSs) of S. pombe, which when cloned into plasmids, support plasmid replication. Three of the proteins have been cloned and sequenced. Two of the proteins, which are called ARS binding protein 1 (abp1) and centromere binding protein B homologue (Cbh), are highly homologous to one another and both possess significant homology to the mammalian centromere DNA binding protein, CENP-B. The protein Abp2 shares no homology to any known protein but contains a motif characteristic of AT-rich DNA binding proteins. The protein Cbh1 has been shown to bind to S. pombe centromeric DNA and appears to play a functional role in stabilizing chromosomes in S. pombe. The investigators plan to further characterize these proteins regarding their specificity in binding to S. pombe centromeric DNA sequences, determine their nuclear localization, utilize them as means to identify other centromere proteins and examine their role in centromeric DNA transactions and functions.